All cellular organisms rely on a multi-protein molecular machine named the replisome to duplicate their genomes. At replication origins, assembly of replisomes for chromosomal DNA replication is a highly regulated event that is coordinated with the cell cycle. In Escherichia coli, DnaA protein initiates chromosomal DNA replication by orchestrating a step-wise process that leads to two DNA helicases bound to each parental DNA strand, which is necessary for bidirectional fork movement from the chromosomal origin. Other components of the replisome then assemble at the newly formed replication fork. Duplication of the genome follows. The recruitment of the replicative helicase to the chromosomal origin is a critical step during the initiation stage of DNA replication. The long-term objective of this research is to understand the molecular mechanism of recruitment and activation of DnaB, the replicative helicase of Escherichia coli, via genetic, biochemical and molecular biological methods. Because all free-living organisms use a similar mechanism of initiation, these studies should provide insight into how this event occurs in higher organisms.